This invention relates to a mass spectrometry system and to a method of mass spectrometric analysis.
Mass spectrometric analysis includes a method of ionizing a sample and directly analyzing the ionized sample (MS analysis) and a method of selecting a specified sample ion (parent ion) according to mass thereof, dissociating the sample ion to form a dissociated ion, and subjecting the dissociated ion to mass spectrometric analysis which is called tandem mass analysis. The tandem method has a function of carrying out dissociation and mass analysis in multi-stage, namely, for example, first selecting out an ion having a specified mass-to-charge ratio (precursor ion) from the dissociated ions, further dissociating the precursor ion, and subjecting the dissociated ion to mass analysis (n-the stage measurement, hereinafter referred to as MSn).
For quantitatively analyzing samples small in quantity and high in the impurity content, a combined system of chromatography and mass analyzer is used. According to this system, a sample to be quantitatively analyzed is separated by time based on the difference in the degree of adsorption to a chromatographic column, or the like, and separated by mass by means of a mass analyzer. In cases of sugar chain isomers or compounds consisting of combination of two different amino acids equal in mass to each other, such materials cannot be separated by mass. However, most of such materials can be separated by time in chromatography according to the difference in chemical properties or physical properties.
Identification of peptides is carried out by a method of using data base search or by a method of reading out the amino acid sequence from the peak distances in the mass spectrometric data. Both these methods are carried out as an after-treatment. The spectral information which has been obtained is insufficient in amount, therefore, it is necessary to collect the data again. Accordingly, this method has not been useful for analyzing quite minute samples, such as disease-formed proteins.
Japanese Patent Kokai 2000-266737 (patent document 1) discloses a method of analyzing the object by comparing the retention times in the sample-separating part and mass spectrum data of the object with those of known substance. However, these treatments are all after treatments. Further, although the comparison with the data of known substance makes it possible to judge that the analyzed sample is an unknown substance, identification of the analyzed sample is difficult to carry out based on such a method.
J. L. Meek, Proc. Natl. Acad. Sci. USA 77, 1632 (1980) (non patent document 1) indicates that, in the case of peptides, retention time can be predicted from the construction of peptide-forming amino acids and the terminal groups. The predicted retention time of a peptide can be calculated based on the sum of the retention time-coefficients of the peptide-forming amino acids and the terminal groups and the elution time of the un-retained compound.
It is an object of this invention to solve a problem that, in the existing mass spectrometry system, whether or not the obtained information is sufficient for analyzing a substance (particularly proteins, sugar chains, etc.) cannot be judged within the actual time period of measurement.
According to the conventional method of mass spectrometry, the species of ion to be subjected to analysis MSn has been determined from the dissociation spectrum of (n−1)th stage (MSn−1), based on the knowledge of the measuring staff. Accordingly, the measurement of MSn has taken a long period of time, so that the spectrometric analysis has usually been carried out only to the stage of n=2. At the stage of n=2, the spectrometric informations necessary for identification have often been unobtainable, and it is difficult in such cases to identify an unknown protein which requires more informations for identification.
If the number of amino acid residues constituting a peptide chain is taken as K and the kind of amino acids is taken as 20, the number of amino acid sequences which can be thought out becomes 20K. If chemical modification of the amino acid side chains is taken into consideration in addition to the above, the number becomes further greater. Such cases include a number of cases where two amino acids are combined together to form an amino acid of which mass coincides with the combined amino acids. Thus, in some states of dissociation of amino acids, it is difficult to distinguish the cases in the term of mass.
According to the data base searching which is a known technique, it is usual to compare the spectrometric data obtained from the 2-th stage of mass analysis with the data base and the degree of coincidence is investigated. Since the data stored in the data base are the second stage mass analysis data for a known substance, identification of unknown substance is impossible. Further, since the quantity of the data accumulated in the data base is huge, there is a high possibility of picking up a number of false positive nominees. According to the de novo peptide sequence method which is a well known method, mass of amino acid is calculated from the peak-peak distance in the dissociation spectrum of n-stage (n≧2), and based on the calculated mass, the amino acid sequence is predicted. Since in this method amino acid sequence is predicted by the use of m/z only, there is a possibility of referring to an enormous number of nominees. Even if the right sequence is involved in such nominees, this method is not adequate from the viewpoint of accuracy of identification. In both the above-mentioned methods, a number of false positive nominees are enumerated, and the work of drawing out the correct answer therefrom requires very much labor and experience.
J. Mass Spectrum. 35, 1399–1406 (2000) (non patent document 2) indicates that, in a case where mobile proton (H+ freely movable between amino acids) is absent (a case that (the number of basic amino acids contained in a peptide)≧(valency number of peptide)) and the peptide contains acidic amino acids such as aspartic acid, glutamic acid and the like, an intense peak of selective dissociation appears in the C-terminal side of acidic amino acid. In this case, the peaks of breakage between other amino acids are very low in intensity, so that it is difficult to identify the object of measurement in a high accuracy.
It is an object of this invention to provide an apparatus for mass spectrometric analysis with which structure and construction of the object of measurement in a high efficiency and accuracy.